*2.1. Leishmania Infantum Culture and Protein Extraction*

*L. infantum* JPCM5 strain parasites were grown at 26 ◦C in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 15% of heat inactivated fetal calf serum (Biowest SAS, Nuaillé, France). Promastigote cultures were initiated at 1 × 10<sup>6</sup> parasites/mL and harvested at the mid-logarithmic phase (1–2 × 10<sup>7</sup> parasites/mL). Around 1–2 × 10<sup>8</sup> parasites were collected and washed twice with phosphate-buffered saline (PBS); finally, parasites were suspended by pipetting in 300 µL of a RIPA (RadioImmunoPrecipitation Assay) lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) in the presence of EDTA-free Easy Pack Protease inhibitor (Roche, Diagnostics, Mannheim, Germany). After 6 cycles (30 s pulse/30 s pause) of sonication in a bath at 4 ◦C, samples were incubated for 90 min at 4 ◦C, and, afterwards, protein lysates were centrifuged at 14,000 g for 30 min. The supernatant was collected and used for proteomics analyses.

## *2.2. In-Gel and In-Solution Digestion of Samples by Trypsin and Chymotrypsin*

For the in-gel digestion of proteins, samples were mixed with an equal volume of a 2× Laemmli buffer and loaded onto 1.2-cm wide wells of a conventional SDS-PAGE gel (0.75 mm-thick, 4% polyacrylamide stacking-gel, and 10% polyacrylamide resolving-gel). The electrophoresis was stopped as soon as the electrophoretic front entered 3 mm into the resolving gel, so the proteins became concentrated in the stacking/resolving gel interface. After Coomassie staining, the protein-containing

gel was cut into small pieces (2 × 2 mm cubes) and placed into a microcentrifuge tube, as described elsewhere [24]. The gel pieces were destained in acetonitrile:water (ACN:H2O, 1:1), reduced and alkylated (disulfide bonds from cysteinyl residues were reduced with 10 mM dithiothreitol (DTT) for 1 h at 56 ◦C, and then thiol groups were alkylated with 10 mM iodoacetamide for 1 h at room temperature in darkness), and digested in situ with sequencing grade trypsin (Promega, Madison, WI) or chymotrypsin (Roche Diagnostics), as described by Shevchenko et al. [25], with minor modifications. The gel pieces were shrunk by removing all liquid using sufficient ACN. Acetonitrile was pipetted out, and the gel pieces were dried in a speedvac. The dried gel pieces were re-swollen in 100 mM Tris-HCl and 10 mM CaCl<sup>2</sup> at pH 8 with 60 ng/µL trypsin or chymotrypsin at a 5:1 protein:enzyme (*w*/*w*) ratio. The tubes were kept on ice for 2 h and incubated at 37 ◦C (trypsin) or 25 ◦C (chymotrypsin) for 12 h. Digestion was stopped by the addition of 1% trifluoroacetic acid (TFA). Whole supernatants were dried down and then desalted onto OMIX C18 pipette tips (Agilent Technologies, Santa Clara, CA, USA) before the MS analysis.

Additionally, in-solution digestion was performed as described elsewhere [26]. After the denaturation of proteins with an 8 M urea, the protein sample was reduced and alkylated: disulfide bonds from cysteinyl residues were reduced with 10 mM DTT for 1 h at 37 ◦C, and then thiol groups were alkylated with 50 mM iodoacetamide for 1 h at room temperature in darkness. The sample was diluted to reduce urea concentration below 1.4 M and digested using sequencing-grade trypsin (Promega, Madison, WI, USA) or chymotrypsin (Roche Diagnostics) overnight at 37 ◦C (trypsin) or 25 ◦C (chymotrypsin) using a 1:20 (*w*/*w*) enzyme/protein ratio. Digestion was stopped by the addition of 1% TFA. Whole supernatants were dried down and then desalted onto OMIX C18 pipette tips (Agilent Technologies) before the MS analysis.
